Conjugates of 1,4,7-triazacyclononanes, dinuclear metal complexes of such conjugates, and methods of use for both 1,4,7-triazacyclononanes and conjugates

ABSTRACT

Conjugates of 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropanes with a variety of conjugating members are used in the formation of dinuclear metal complexes which bind to phosphate esters. By virtue of their conjugated forms, the complexes are incorporated into chromatographic media, affinity binding reagents, and dyes, which make the complexes useful in a wide range of assays, separations, and purifications. In addition, dinuclear metal complexes of 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropanes that are not so conjugated are used in the detection of phosphate esters of biological species by either MALDI-TOF mass spectrometry or by dye displacement.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplications No. 61/314,431, filed Mar. 16, 2010, and No. 61/368,106,filed Jul. 27, 2010. The contents of both such provisional patentapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Phosphorylation and dephosphorylation are metabolically significantreactions in physiological systems. These reactions occur at hydroxylgroups of certain proteins, at nitrogen atoms of certain other proteins,and at carboxyl groups of still more proteins, in each case having aneffect on the activity and function of these proteins in vivo. Certainphosphorylation/-dephosphorylation reactions are mediated by proteinkinases and phosphatases and their occurrences provide an indication ofthe activity of these enzymes. Abnormalities inphosphorylation/dephosphorylation reactions have been implicated in cellcarcinogenesis, in allergic disorders, and in Alzheimer's disease.Protein phosphorylation is an essential process in all living cells, forsignal transduction, apoptosis, proliferation, differentiation, andmetabolism. Methods for the determination of the phosphorylation statusof proteins are thus valuable in exploring the molecular origins ofdiseases and in the design of new drugs.

Certain metalloenzymes that contain two metal ions are known to catalyzedephosphorylation reactions, in particular the hydrolysis of phosphatediesters. Studies of the mechanism by which these reactions occur haveinvolved the use of synthetic organic ligands that form dimetalliccomplexes and perform the same catalytic function. Most of thesesynthetic ligands were found to have low solubility in water, however,which limited their utility in laboratory studies. To remove thisobstacle, a water-soluble ligand,1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane, has been developed.Disclosures of Zn(II) complexes of this ligand and its use in thehydrolysis of RNA are published by Iranzo, O., et al., “Cooperativitybetween Metal Ions in the Cleavage of Phosphate Diesters and RNA byDinuclear Zn(II) Catalysts,” Inorganic Chemistry 42(24), 7737-7746(2003), and Iranzo, O., et al., “Physical and Kinetic Analysis of theCooperative Role of Metal Ions in Catalysis of Phosphodiester Cleavageby a Dinuclear Zn(II) Complex,” J. Am. Chem. Soc. 125(7), 1988-1993(2003). Further studies of this molecule and of the transition statethat it forms with the phosphate esters are reported by Yang, M.-Y., etal., “Substrate specificity for catalysis of phosphodiester cleavage bya dinuclear Zn(II) complex,” Chem. Commun. 2003, 2832-2833, and Yang,M.-Y., et al., “A transition state analog for phosphate diester cleavagecatalyzed by a small enzyme-like metal ion complex,” BioorganicChemistry 35, 366-374 (2007).

SUMMARY OF THE INVENTION

In part, the present invention resides in conjugates of substituted orunsubstituted 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropanes witha variety of conjugating members, each conjugate capable of forming adimetallic complex in the same manner as the complexes disclosed in thereports cited above. The invention also resides in functionalized1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropanes, substituted orunsubstituted, the functional groups of which are reactive with aconjugating member. The substituted or unsubstituted1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropanes are referred to forconvenience herein as “ligands,” and the conjugates are those ligandsthat are conjugated with a conjugating member that serves one of avariety of additional functions as described below. The inventionfurther resides in the dinuclear metal complexes of these conjugates,and in various uses of these complexes other than in the hydrolysis ofphosphate diesters. The utility of each complex arises from theparticular conjugating member used in forming the conjugate, inconjunction with the phosphate-binding activity of the two metal ions.

The present invention further resides in methods for detecting phosphateesters in a sample by MALDI-TOF (Matrix-Assisted LaserDesorption/Ionization Time-of-Flight) mass spectrometry, using eitherdinuclear metal complexes of the ligands or dinuclear metal complexes ofthe conjugates of the ligands, by mixing such a complex with the samplein a suitable matrix to form positively charged dinuclear metalcomplexes of the phosphate esters. When the mass spectrometry scan isrun on the sample and matrix, the differentiation of the positivelycharged dinuclear metal complexes of phosphate esters in the sample fromuncomplexed components is increased due to the additional mass impartedby the ligand in the complex as well as a change in the charge of thecomplex. Ligands that are not conjugated are of particular interest inthis aspect of the invention.

A still further aspect of the invention resides in methods for detectingphosphate esters in a sample by dye displacement, again using eitherdinuclear metal complexes of the ligands or dinuclear metal complexes ofthe conjugates of the ligands. In this aspect of the invention, thedinuclear metal complexes are further complexed with a chromophoriccompound that has two key characteristics—(1) the chromophoric compoundhas a lower affinity to the metal-charged ligand (or conjugate of theligand) than do the phosphate esters, i.e., the phosphate esters bindmore strongly to the ligand and will displace the chromophoric compoundin the complex upon contact; and (2) the chromophoric compound has adifferent absorbance or fluorescence spectrum when bound than whenunbound. One example of a chromophoric dye that meets this descriptionis pyrocatechol violet. The spectral change that occurs as a result ofthe displacement of the chromophoric compound from the complex by thephosphate esters thus serves as an indication of the presence of thephosphate esters in the sample. In this aspect of the invention as well,the use of an unconjugated ligand is of particular interest.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The ligands, which are substituted or unsubstituted1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane, are represented bygeneric Formula (I):

In Formula (I), the groups R¹, R², R³, R⁴, R⁵, and R⁶ are the same ordifferent (some the same and others different) and are either H or alower alkyl group. The expression “lower alkyl” is defined as an alkylgroup of six carbon atoms or less. Preferred groups for R¹ through R⁶are either H, C₁-C₃ alkyl, or combinations selected from H and C₁-C₃alkyl, and the most preferred is H. The term “substituted” in thiscontext denotes that one or more of R¹, R², R³, R⁴, and R⁶ is an alkylgroup rather than an H atom.

Ligands that are described herein as not being conjugated are those inwhich R¹, R², R³, R⁴, R⁵, and R⁶ are the same or different and each iseither H or a lower alkyl group. The conjugates of the ligands are thosecompounds of Formula (I) in which a conjugating member occupies any ofthe positions indicated by R¹, R², R³, R⁴, R⁵, and R⁶, through a linkinggroup joining the conjugating member to the remainder of the structure.Any such R groups that do not represent the conjugating member areeither H or a lower alkyl group, as indicated above. For the conjugates,one or more of R¹ through R⁶ is thus -L-R⁷, where L represents thelinking group and R⁷ is the conjugating member, and the remaininggroup(s) of R¹ through R⁶, which are either the same or different (somethe same and others different), are each either H or C₁-C₆ alkyl. Forthe functionalized ligands, one or more of R¹ through R⁶ is -L′, whereL′ represents the unreacted linking group that is reactive with theconjugating member. In the functionalized ligands as in the conjugates,the remaining group(s) of R¹ through R⁶, which are either the same ordifferent (some the same and others different), are each either H orC₁-C₆ alkyl. In preferred functionalized ligands or conjugates, only oneof R¹ through R⁶ is -L′ or -L-R⁷, respectively, and in further preferredligands or conjugates, only R⁵, only R⁶, or only R² is -L′ or -L-R⁷. Ofthe R¹ through R⁶ groups that are not replaced by -L′ or -L-R⁷, suchgroups are preferably either H, C₁-C₃ alkyl, or combinations selectedfrom H and C₁-C₃ alkyl, and the most preferred is H. The conjugatingmember R⁷ is either an acrylamide group, a fluorescent dye, anaffinity-type binding member, or a solid chromatographic support. Thelinking group L′ or L is any conventional linker with two binding sites,one of which forms a covalent bond with the conjugating member R⁷ andthe other forms a covalent bond with the adjacent carbon atom on theremainder of Formula (I). The linking group generally has from 3 to 30atoms selected from C, N, O, P, and S, in addition to hydrogen atomsfilling available valences, and is either cyclic, acyclic, aromatic or acombination of cyclic, acyclic, and aromatic moieties. Examples oflinking groups are amine alkyl linkages, alkenyl linkages, amide oramide-containing linkages, ester or ester-containing linkages, and etheror ether-containing linkages. Preferred linking groups for L′ are—(C₁-C₄ alkyl)-NH₂, —(C₁-C₄ alkyl)-C(═O)—NH₂, —(C₁-C₄ alkyl)-NH—CO₂H,—(C₁-C₄ alkyl)-C(═O)—NH—(C₁-C₄ alkyl)-NH₂, and —(C₁-C₄alkyl)-NH—C(═O)—(C₁-C₄)—NH₂, and particularly preferred are —CH₂CH₂—NH—,—CH₂—C(═O)—NH₂, —CH₂CH₂—NH—CO₂H, —CH₂—C(═O)—NH—CH₂CH₂—NH₂, and—CH₂CH₂—NH—C(═O)—CH₂CH₂—NH₂. Preferred groups for L are (C₁-C₄alkyl)-C(═O)—NH—(C₁-C₄ alkyl)-, —(C₁-C₄ alkyl)-NH—C(═O)— (C₁-C₄alkyl)-NH—, and —(C₁-C₄ alkyl)-NH—, and particularly preferred are—CH₂—C(═O)—NH—CH₂—CH₂—, —CH₂—CH₂—NH—C(═O)—CH₂—CH₂—NH—, and —CH₂—CH₂—NH—.

When R⁷ is an acrylamide group, -L-R⁷ is preferably—CH₂—C(O)—NH—(CH₂)_(n)—NH—C(O)—CH═CH₂ or—(CH₂)₂—NH—C(O)—(CH₂)_(n)—NH—C(O)—CH═CH₂ where n is 1 to 6, and the mostpreferred are —CH₂—C(O)—NH—(CH₂)₂—NH—C(O)—CH═CH₂ and—(CH₂)₂—NH—C(O)—(CH₂)₂—NH—C(O)—CH═CH₂. When this particular -L-R⁷replaces R⁵ and the remaining R's are all H, the conjugate has theformula

Functionalized polyacrylamide gels can be formed from conjugates anddinuclear metal complexes in accordance with this invention where theconjugating member is acrylamide. The functional group in these gels isthe Bimetal complex of the acrylamide-containing conjugate, and theresulting gels can be used for electrophoretic separations of phosphatemonoesters of proteins, peptides, or nucleic acids from thenon-phosphorylated forms of these species. Phosphate monoesters ofproteins, peptides, or nucleic acids within the mixture will migratemore slowly during electrophoresis due to interaction between thephosphate esters and the dimetal complex functional groups on the gels.Phosphorylated components of the mixture can thereby be distinguishedfrom their unphosphorylated counterparts. The functionalized gel isformed by including the dimetal complex in the monomer mixture, whichwill generally also include non-functionalized acrylamide, acrosslinker, and an initiator. Suitable crosslinkers and initiators arewell known in the art. Examples of crosslinkers are bisacrylamide andethylene diacrylate, and examples of initiators are riboflavin, ammoniumpersulphate, and tetramethylethylenediamine (TEMED). The conditions forpolymerization of the monomers and the concentrations of the variouscomponents of the monomer mixture are the same as those used inprocedures of the prior art for the formation of the polyacrylamidegels.

Dinuclear metal complexes of the ligands of Formula (I) for use in thepractice of this invention are those having Formula (II):

In Formula (II), R¹ through R⁶ are as defined above, and M is a divalentmetal. A preferred group of divalent metals is Ca, Zn, Cr, Mn, Fe, Co,Ni, and Cu (all in divalent form). Particularly preferred divalentmetals are Zn (II) and Mn(II).

When used in the various methods of the present invention, the dinuclearmetal complexes of Formula (II) form phosphate association complexes ofFormula (III):

In Formula (III), X is the residue of a peptide, protein, nucleic acid,or in general any species to which the complexes of this invention areto be applied for any of the various purposes stated herein.

When R⁷ is a fluorescent dye, the dye can be any of the variety offluorescent dyes known for use in the labeling of biological species.Examples are:

-   -   fluorescein and fluorescein derivatives such as fluorescein        isothiocyanate,    -   rhodamine derivatives such as tetramethyl rhodamine, rhodamine        B, rhodamine 6G, sulforhodamine B, rhodamine 101 (Texas Red),        and rhodamine 110,    -   4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) and        derivatives thereof,    -   pyrenes and pyrene derivatives such as        8-methoxypyrene-1,3,6-trisulfonic acid,    -   pyridyloxazole derivatives,    -   dapoxyl derivatives,    -   umbelliferone,    -   1-anilino-8-naphthalenesulfonic acid,    -   3,6-disulfonate-4-amino-naphthalimide,    -   tri-, penta-, and heptamethine cyanine dyes, and    -   luminescent transition metal complexes such as        tris(2,2′-bipyridine)ruthenium(II) or cyclometalated complexes        of Ir(III).

When R⁷ is tetramethyl rhodamine, for example, -L-R⁷ is preferably

where the asterisk denotes the site of attachment to the1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane. When this linkerand dye replace R⁵ and R¹ through R⁴ are all H, the resulting conjugatehas the formula

Dinuclear metal complexes in accordance with this invention that areformed from conjugates containing fluorescent dyes can be used for thestaining of gels or blots to identify and quantify phosphate esters,such as phosphoproteins, for example, in samples of biological fluidsand in assay media in general.

When R⁷ is an affinity-type binding member, examples of such bindingmembers are biotin, avidin, streptavidin, antibodies, and antibodyfragments. When R⁷ is biotin, -L-R⁷ is preferably

where the asterisk denotes the site of attachment to the1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane. When thisfunctional group replaces R⁵ and R¹ through R⁴ are all H, the resultingconjugate has the formula

Dinuclear metal complexes in accordance with this invention in which R⁶is an affinity binding member are useful in the detection ofphosphoproteins and other phosphate esters bound to or otherwiseimmobilized on a solid support. The ability of the affinity bindingmember to bind with specificity to a second affinity binding member toform an affinity binding pair allows the attachment of a reporter groupto the complexes through the binding pair, either before or after thecomplexes have associated with the phosphate esters. This allowsdetection by any type of reporter group that can be conjugated to anaffinity binding member. Affinity binding pairs are well known; examplesare biotin and avidin, biotin and streptavidin, and any of variousantibodies or antibody fragments and antigens. Examples or reportergroups are radioactive labels, chemiluminescent labels, and enzymes.Examples of enzymes are horseradish peroxidase, chloramphenicol acetyltransferase, β-galactosidase, alkaline phosphatase, and luciferase. Toillustrate the use of dinuclear metal complexes containing an affinitybinding member, the solid support, such as for example, a blottingmembrane, on which the phosphate esters have been captured is firstcontacted with the complex. If, for example, the conjugating member onthe complex is biotin, the support is contacted with astreptavidin-enzyme conjugate. This is followed by incubation of thesupport with an enzyme substrate and detection of the change, most oftena color change, in the substrate, mediated by the enzyme. Optimalconditions for the contact and incubation are readily apparent to thoseof skill in the art.

When R⁷ is a solid chromatographic support, the term “solid” is usedherein to include semi-solids such as gels, flexible solids such asmembranes and films, and rigid solids such as those used in theformation of incompressible beads, granules, and column or tube walls.Examples of such solids are polyacrylamide, crosslinked siliconpolymers, silica gel, agarose, polyvinyl alcohols, cellulose andnitrocellulose. A chromatographic support bearing the dinuclear metalcomplexes of this invention can be used to extract phosphorylatedcompounds (phosphate esters) from fluids such as biological fluids,assay media, or any fluids containing phosphorylated compounds, forpurposes of purification or enrichment.

Conjugates in accordance with this invention can be prepared byconventional procedures, beginning with 1,4,7-triazacyclononanederivatives with one or more of the secondary amine groups protectedeither with a suitable protecting group or by formation of a1,4,7-triazacyclononane tricyclic orthoamide. Appropriate functionalgroups can be selectively placed in one or more of the unprotected R¹through R⁴ positions. An appropriate functional group can also be placedin the R⁵ position by selection of an appropriate synthetic intermediatethat will join the two 1,4,7-triazacyclononane groups by connecting onering nitrogen from each macrocycle with a three-carbon chain. The choiceof functional group will vary with the choice of linking group and withthe means by which the linking group is attached, such attachment beingachievable by any of the wide range of linking reactions known in theart. Prominent example of functional groups are carboxylic acids oresters of carboxylic acids that can react with an amine functionalityappended to the conjugating member, or an amine group that can reactwith an active carboxylic acid ester or acid chloride, both resulting inthe formation of an amide linkage. Reaction of a carboxylicacid-functionalized ligand with an amine-bearing conjugation partner, asknown in the art, is readily achieved either directly or in the presenceof an activated acid such as an acid chloride, acid anhydride, orsuccinimidyl ester. Reaction of an amine-functionalized ligand with anactivated ester, or acid chloride-bearing conjugation partner, as knownin the art, is readily achieved.

The preparation of functionalized ligands is illustrated by a ligand inwhich a methyl ester occupies the R⁵ position, with all other R-groupsbeing H. An example of such a preparation is one that begins with1,4,7-triazacyclononane and reacts with dimethylformamide dimethylacetal to form 1,4,7-triazacyclononane tricyclic orthoamide, which isthen reacted with methyl 3,5-dibromolevulinate to form methyl3,5-bis(4-formyl-1,4,7-triazonan-1-yl)-4-oxopentanoate. The latter isthen reacted with sodium borohydride to form methyl3,5-bis(4-formyl-1,4,7-triazonan-1-yl)-4-hydroxypentanoate, which isthen reacted with hydrochloric acid to produce methyl4-hydroxy-3,5-di(1,4,7-triazonan-1-yl)pentanoate. This sequence ofreactions is shown below:

Variations of this procedure to place the methyl ester group at otherlocations on the molecule and to obtain the functionalized ligand withadditional substitutions will be apparent to those skilled in organicchemical syntheses. One example is shown below, which places the aminegroup and starts with 1,4,7-triazacyclononane that is protected at two Natoms with butoxycarbonyl groups is reacted with 1,3-dibromo-2-propanolto form di-tert-butyl7-(3-bromo-2-hydroxypropyl)-1,4,7-triazonane-1,4-dicarboxylate (Step 1).Separately, unprotected 1,4,7-triazacyclononane is reacted withN,N-dimethylformamide dimethyl acetal to form the correspondingtricyclic structure, which is then reacted with tert-butyl(2-bromoethyl)carbamate and hydrolyzed, yielding tert-butyl(2-(4-formyl-1,4,7-triazanon-1-yl)-3-)1,4,7-triazonan-1-yl)ethylcarbamate. The two products are then reacted to form1-(4-(2-aminoethyl)-1,4,7-triazonan-1-yl)-3-(1,4,7-triazonan-2-yl)propan-2-olfollowing deprotection (Step 3).

Step 1:

Step 2:

Step 3:

A further example is shown below, starting with the hydration ofN-(3-butynyl)phthalamide to N-(3-oxobutyl)phthalamide. This is followedby reaction with bromine, then with a Wittig reagent, then withperoxyacetic acid, then with 1,4,7-triazacyclononane protected at two Natoms, followed by deprotection to form2-(1,4,7-trazonan-1-yl)methyl)-4-1-(1,4,7-triazonan-1-yl)butan-2-ol.

Complexation of either the ligands or the conjugates for use in thepresent invention can be performed on the ligand, on the functionalizedligand prior to conjugation, or on the conjugate itself. In either case,complexation can be achieved by combining an appropriate salt of themetal, for example Zn(NO₃)₂ where a Zn complex is to be formed, with thehydrobromide or hydrochloride salt of the ligand at an appropriate molarratio, and adjusting the pH to 6.5-7.0.

In the claims appended hereto, the term “a” or “an” is intended to mean“one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is optional and not excluded. All patents, patent applications,and other published reference materials cited in this specification arehereby incorporated herein by reference in their entirety. Anydiscrepancy between any reference material cited herein or any prior artin general and an explicit teaching of this specification is intended tobe resolved in favor of the teaching in this specification. Thisincludes any discrepancy between an art-understood definition of a wordor phrase and a definition explicitly provided in this specification ofthe same word or phrase.

What is claimed is:
 1. A compound having the formula

in which: one or more of R¹ through R⁶ is -L′ in which L′ is a linking group reactive with a conjugating member selected from the group consisting of an acrylamide group, a fluorescent dye, an affinity-type binding member, and a solid chromatographic support, to form a conjugate of said compound with said conjugating member; and the remainder of R¹ through R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl.
 2. The compound of claim 1 wherein L′ is a member selected from the group consisting of —(C₁-C₄ alkyl)-NH₂, —(C₁-C₄ alkyl)-C(═O)—NH₂, —(C₁-C₄ alkyl)-NH—CO₂H, —(C₁-C₄ alkyl)-C(═O)—NH—(C₁-C₄ alkyl)-NH₂, and —(C₁-C₄ alkyl)-NH—C(═O)—(C₁-C₄)—NH₂.
 3. The compound of claim 1 wherein L′ is a member selected from the group consisting of —CH₂CH₂—NH—, —CH₂—C(═O)—NH₂, —CH₂CH₂—NH—CO₂H, —CH₂—C(═O)—NH—CH₂CH₂—NH₂, and —CH₂CH₂—NH—C(═O)—CH₂CH₂—NH₂.
 4. A compound having the formula

in which: one or more of R¹ through R⁶ is -L-R⁷ in which L is a linking group and R⁷ is a member selected from the group consisting of an acrylamide group, a fluorescent dye, an affinity-type binding member, and a solid chromatographic support; and the remainder of R¹ through R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl.
 5. The compound of claim 4 wherein R⁵ is -L-R⁷, and R¹, R², R³, R⁴, and R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl.
 6. The compound of claim 4 wherein R⁵ is -L-R⁷, and R⁵, R², R³, R⁴, and R⁶ are H.
 7. The compound of claim 4 wherein R² is -L-R⁷, and R¹, R³, R⁴, R⁵, and R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl.
 8. The compound of claim 4 wherein R² is -L-R⁷, and R¹, R³, R⁴, R⁵, and R⁶ are H.
 9. The compound of claim 4 wherein R⁶ is -L-R⁷, and R¹, R², R³, R⁴, and R⁵ are independently selected from the group consisting of H and C₁-C₆ alkyl.
 10. The compound of claim 4 wherein R⁶ is -L-R⁷, and R¹, R², R³, R⁴, and R⁵ are H.
 11. The compound of claim 4 wherein L is a member selected from the group consisting of —CH₂—C(═O)—NH—(C₁-C₄ alkyl)-, —CH₂—C(═O)—NH—(C₁-C₄ alkyl)-NH—, and —(C₁-C₄ alkyl)-NH—.
 12. The compound of claim 4 wherein L is a member selected from the group consisting of —CH₂—C(═O)—NH—CH₂—CH₂—, —CH₂—C(═O)—NH—CH₂—CH₂—NH—, and —C₂H₅—NH—.
 13. The compound of claim 4 wherein R⁷ is an acrylamide group.
 14. The compound of claim 4 wherein R⁵ is —CH₂—C(═O)—NH—CH₂—CH₂—NH—C(═O)—CH═CH₂, and R¹, R², R³, R⁴, and R⁶ are H.
 15. The compound of claim 4 wherein R⁵ is —CH₂—CH₂—NH—C(═O)—CH═CH₂, and R¹, R², R³, R⁴, and R⁶ are H.
 16. The compound of claim 4 wherein R² is —CH₂—CH₂—NH—C(═O)—CH═CH₂, and R¹, R³, R⁴, R⁵, and R⁶ are H.
 17. The compound of claim 4 wherein R⁶ is —CH₂—CH₂—NH—C(═O)—CH═CH₂, and R¹, R², R³, R⁴, and R⁵ are H.
 18. The compound of claim 4 wherein R⁷ is a fluorescent dye.
 19. The compound of claim 4 wherein R⁷ is biotin.
 20. The compound of claim 4 wherein R⁷ is a solid chromatographic support.
 21. A compound having the formula

in which: one of R¹ through R⁶ is -L-R⁷ in which L is a linking group and R⁷ is a member selected from the group consisting of an acrylamide group, a fluorescent dye, an affinity-type binding member, and a solid chromatographic support; the remainder of R¹ through R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl; and M is a divalent metal.
 22. The compound of claim 21 wherein M is a member selected from the group consisting of Ca, Zn, Cr, Mn, Fe, Co, Ni, and Cu.
 23. The compound of claim 21 wherein M is a member selected from the group consisting of Zn and Mn.
 24. The compound of claim 21 wherein R⁵ is -L-R⁷, and R¹, R², R³, R⁴, and R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl.
 25. The compound of claim 21 wherein R⁵ is -L-R⁷, and R¹, R², R³, R⁴, and R⁶ are H.
 26. The compound of claim 21 wherein R² is -L-R⁷, and R¹, R³, R⁴, R⁵, and R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl.
 27. The compound of claim 21 wherein R² is -L-R⁷, and R¹, R³, R⁴, R⁵, and R⁶ are H.
 28. The compound of claim 21 wherein R⁶ is -L-R⁷, and R¹, R², R³, R⁴, and R⁵ are independently selected from the group consisting of H and C₁-C₆ alkyl.
 29. The compound of claim 21 wherein R⁶ is -L-R⁷, and R¹, R², R³, R⁴, and R⁵ are H.
 30. The compound of claim 21 wherein L is a member selected from the group consisting of —CH₂—C(═O)—NH—(C₁-C₄ alkyl)-, —CH₂—C(═O)—NH—(C₁-C₄ alkyl)-NH—, and —(C₁-C₄ alkyl)-NH—.
 31. The compound of claim 21 wherein L is a member selected from the group consisting of —CH₂—C(═O)—NH—CH₂—CH₂—, —CH₂—C(═O)—NH—CH₂—CH₂—NH—, and —C₂H₅—NH—.
 32. The compound of claim 21 wherein R⁷ is an acrylamide group.
 33. The compound of claim 21 wherein R⁵ is —CH₂—C(═O)—NH—CH₂—CH₂—NH—C(═O)—CH═CH₂, and R¹, R², R³, R⁴, and R⁶ are H.
 34. The compound of claim 21 wherein R⁵ is —CH₂—CH₂—NH—C(═O)—CH═CH₂, and R¹, R², R³, R⁴, and R⁶ are H.
 35. The compound of claim 21 wherein R² is —CH₂—CH₂—NH—C(═O)—CH═CH₂, and R¹, R³, R⁴, R⁵, and R⁶ are H.
 36. The compound of claim 21 wherein R⁶ is —CH₂—CH₂—NH—C(═O)—CH═CH₂, and R¹, R², R³, R⁴, and R⁵ are H.
 37. The compound of claim 21 wherein R⁷ is a fluorescent dye.
 38. The compound of claim 21 wherein R⁷ is biotin.
 39. The compound of claim 21 wherein R⁷ is a solid chromatographic support.
 40. A method for the preparation of a separation medium for use in electrophoresis of phosphorylated species, said method comprising polymerizing a monomer mixture comprising acrylamide, a crosslinker, an initiator, and a compound having the formula

in which: one of R¹ through R⁶ is -L-R⁷ in which L is a linking group and R⁷ is an acrylamide group; the remainder of R¹ through R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl; and M is a divalent metal.
 41. A method of staining phosphorylated compounds immobilized on a solid support or in a gel with a fluorescent dye, said method comprising contacting said solid support or gel with a compound having the formula

in which: one of R¹ through R⁶ is -L-R⁷ in which L is a linking group and R⁷ is a fluorescent dye; the remainder of R¹ through R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl; and M is a divalent metal.
 42. A method for detecting a phosphorylated compound immobilized on a solid support or in a gel, said method comprising: (A) contacting said solid support or gel with a compound having the formula

in which: one of R¹ through R⁶ is -L-R⁷ in which L is a linking group and R⁷ is a first binding member; the remainder of R¹ through R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl; and M is a divalent metal; and (B) subsequent to (A), contacting said solid support or gel with a conjugate of (i) a second binding member that binds to said first binding member upon contact through affinity type binding and (ii) a reporter group.
 43. A method for extraction of phosphorylated species from a liquid solution, said method comprising passing said liquid solution through an extraction medium comprising the following compound conjugated to a solid support:

in which: one of R¹ through R⁶ is -L-* in which L is a linking group and * is a site of attachment to said solid support; the remainder of R¹ through R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl; and M is a divalent metal.
 44. A method of detecting phosphate esters in a sample, said method comprising: (A) forming a mixture of said sample and a compound of the formula:

in which R¹ through R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl, and M is a divalent metal, to form complexes of any such phosphate esters present in said sample with said compound, and (B) scanning said mixture by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to detect said complexes and thereby differentiate said phosphate esters in said sample from species in said sample other than phosphate esters.
 45. A method of detecting phosphate esters in a sample by dye displacement, said method comprising: (A) forming a mixture of said sample and a complex of a metal-charged ligand and a chromophoric compound, wherein said metal-charged ligand has the formula:

in which R¹ through R⁶ are independently selected from the group consisting of H and C₁-C₆ alkyl, and M is a divalent metal, and said chromophoric compound has a binding affinity of a first magnitude to said metal-charged ligand and phosphate esters have a binding affinity of a second magnitude to said metal-charged ligand, said first magnitude being lower than said second magnitude, and said chromophoric compound has a first emission spectrum when bound to said metal-charged ligand and a second emission spectrum when unbound, said first and second emission spectra being distinguishable from each other, (B) detecting emission spectra of said mixture as an indication of the presence or absence of said phosphate esters in said sample. 